Ligand-guided-selection method for screening antigen-specific ligands

Abstract

A Ligand-guided-Selection (LIGS) method for identifying highly specific aptamers against a predetermined antigen of a target is provided. LIGS uses a stronger and highly specific bivalent binder (e.g. an antibody) interacting with its cognate antigen to displace specific aptamers from an enriched SELEX pool. Elution of the displaced aptamers provides aptamers that are specific to the predetermined antigen.

What is claimed is: 
     
       1. A Ligand-guided-Selection method for screening ligands that are specific to a predetermined antigen, the method comprising sequential steps of:
 forming a ligand-cell complex by exposing a culture of target cells to a library of ligands that is at least partially enriched, wherein cells in the culture of target cells each have the predetermined antigen; 
 treating the ligand-cell complex with a predetermined ligand that is specific to the predetermined antigen, the predetermined ligand displacing ligands that are bound to the predetermined antigen to form displace ligands, the step of treating leaving non-displaced ligands bound to the ligand-cell complex, wherein the predetermined ligand is an antibody; 
 eluting the displaced ligands; and 
 amplifying the displaced ligands; wherein the step of amplifying the displaced ligands comprises a polymerase chain reaction (PCR). 
 
     
     
       2. The method as recited in  claim 1 , wherein the antibody has a molecular weight between 65 kDa and 150 kDa. 
     
     
       3. The method as recited in  claim 1 , further comprising treating the ligand-cell complex with an off-target antibody that binds at an antigen other than the predetermined antigen. 
     
     
       4. The method as recited in  claim 1 , wherein the culture of target cells is washed to remove free ligands prior to the step of forming and the antibody is present in a molar excess relative to the predetermined antigen. 
     
     
       5. The method as recited in  claim 1 , wherein the culture of target cells is not washed prior to the step of forming, thus leaving free ligands present during the step of forming and the antibody is present in a molar excess relative to the predetermined antigen. 
     
     
       6. The method as recited in  claim 1 , wherein free ligands are present during the step of forming. 
     
     
       7. The method as recited in  claim 1 , further comprising a step of removing free ligands by washing prior to the step of forming. 
     
     
       8. The method as recited in  claim 1 , wherein the library of ligands has a plurality of aptamers, each having a dissociation constant (K d ), wherein the step of forming uses a concentration of each aptamer equal to or below its dissociation constant (K d ). 
     
     
       9. The method as recited in  claim 1 , wherein the library of ligands has a plurality of aptamers, each having a dissociation constant (K d ), wherein the step of forming uses a concentration of each aptamer equal to or below half of its dissociation constant (K d ). 
     
     
       10. The method as recited in  claim 1 , wherein the antibody has a molecular weight greater than 150,000 g per mole. 
     
     
       11. The method as recited in  claim 1 , wherein the antibody is present in at least a five-fold molar excess relative to the predetermined antigen. 
     
     
       12. The method as recited in  claim 1 , wherein the antibody is present in at least a ten-fold molar excess relative to the predetermined antigen. 
     
     
       13. A Ligand-guided-Selection method for screening ligands that are specific to a predetermined antigen, the method comprising sequential steps of:
 constructing a library of ligands that is at least partially enriched by:
 exposing a target cell line to an aptamer library and permitting at least some aptamers to bind to the target cell line, thereby forming bound aptamers; 
 removing aptamers that do not bind to the target cell line; 
 eluting the bound aptamers, thereby forming eluted aptamers; 
 amplifying the eluted aptamers that are specific to the cell line, thereby forming the library of ligands; 
 
 forming a ligand-cell complex by exposing a culture of target cells to the library of ligands, wherein cells in the culture of target cells each have the predetermined antigen; 
 treating the ligand-cell complex with an antibody that is specific to the predetermined antigen, the antibody displacing ligands that are bound to the predetermined antigen to form displace ligands, the step of treating leaving non-displaced ligands bound to the ligand-cell complex; 
 eluting the displaced ligands; 
 amplifying the displaced ligands using polymerase chain reaction (PCR). 
 
     
     
       14. The method as recited in  claim 13 , further comprising:
 performing a sequence alignment on first ligands from the library of ligands and second ligands from the displaced ligands; 
 identifying, based on the sequence alignment, a common sequence of nucleotides. 
 
     
     
       15. The method as recited in  claim 14  wherein the step of identifying identifies the common sequence of nucleotides by finding at least four repeats of the common sequence within the first ligands from the library of ligands and second ligands from the displaced ligands. 
     
     
       16. A method for selecting aptamers using an antibody-capped cell Systematic Evolution of Ligands by EXponential enrichment process, the method comprising steps of:
 exposing an antibody-capped cell to a plurality of different aptamers and permitting at least some aptamers to bind to the antibody-capped cell to form bound aptamers, wherein the antibody-capped cell has been pretreated with a predetermined antibody that caps an antigen; 
 eluting unbound aptamers; and 
 amplifying the unbound aptamers. 
 
     
     
       17. The method as recited in  claim 16 , wherein the plurality of different aptamers are produced by:
 exposing a target cell to an aptamer library; 
 permitting at least some aptamers in the aptamer library to bind to the target cell to form bound aptamers; 
 eluting unbound aptamers; 
 eluting the bound aptamers from the target cell to produce the plurality of different aptamers.